Dual functions of Rift Valley fever virus NSs protein: inhibition of host mRNA transcription and post-transcriptional downregulation of protein kinase PKR - PubMed (original) (raw)

Dual functions of Rift Valley fever virus NSs protein: inhibition of host mRNA transcription and post-transcriptional downregulation of protein kinase PKR

Tetsuro Ikegami et al. Ann N Y Acad Sci. 2009 Sep.

Abstract

Rift Valley fever virus (RVFV), which belongs to the genus Phlebovirus, family Bunyaviridae, is a negative-stranded RNA virus carrying a single-stranded, tripartite RNA genome. RVFV is an important zoonotic pathogen transmitted by mosquitoes and causes large outbreaks among ruminants and humans in Africa and the Arabian Peninsula. Human patients develop an acute febrile illness, followed by a fatal hemorrhagic fever, encephalitis, or ocular diseases. A viral nonstructural protein, NSs, is a major viral virulence factor. Past studies showed that NSs suppresses the transcription of host mRNAs, including interferon-beta mRNAs. Here we demonstrated that the NSs protein induced post-transcriptional downregulation of dsRNA-dependent protein kinase (PKR), to prevent phosphorylation of eIF2alpha and promoted viral translation in infected cells. These two biological activities of the NSs most probably have a synergistic effect in suppressing host innate immune functions and facilitate efficient viral replication in infected mammalian hosts.

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Figures

Figure 1

Figure 1

Effects of ActD on the replication of MP-12 and MP-12 lacking the NSs gene. The Fig is adapted from Ikegami et al.[26] (A) Schematic representations of the S segments of MP-12 and rMP12-rLuc. (B) Type I IFN-deficient VeroE6 cells were mock-treated or independently infected with MP-12 and rMP12-rLuc at an moi of 3, immediately treated with ActD (5 μg/ml) or left untreated, and culture fluids were harvested at 16 h.p.i. Virus titers of MP-12 and rMP12-rLuc were measured by a plaque assay. The virus replication of rMP12-rLuc was significantly reduced in the presence of ActD (*p<0.001; Student's _t_-test). Data are expressed as mean +/- standard deviation of three independent experiments.

Figure 2

Figure 2

Status of eIF2α phosphorylation in rMP12-rLuc-infected cells and MP-12-infected cells in the presence of transcriptional inhibitors. The Fig is adapted from Ikegami et al.[26] VeroE6 cells were mock infected (M) or infected with rMP12-rLuc (A) or MP-12 (B) at an moi of 3, and then immediately treated with ActD or left untreated (No drug). Samples were harvested at the indicated time points post infection for Western blot analysis. RVFV N protein, NSs proteins, phosphorylated eIF2α, total eIF2α, and β-actin are shown by arrowheads. The data are representative of three independent experiments.

Figure 3

Figure 3

Role of PKR in eIF2α phosphorylation in infected cells under transcriptional suppression. The Fig is adapted from Ikegami et al.[26] (A) Schematic representations of S segments of MP-12 and rMP12-PKRΔE7. (B) VeroE6 cells were independently infected with MP-12, rMP12-rLuc and rMP12-PKRΔE7 at an moi of 3, or were mock infected. Cells were immediately treated with ActD (Act) or 50 μg/ml of α-amanitin (Ama), or were untreated. Cell extracts were prepared at 16 h.p.i. for Western blot analysis. Western blot analysis showing the accumulation of eIF2α, phosphorylated eIF2α, N protein, NSs protein, Flag-PKRΔE7 and β-actin in infected VeroE6 cells.

Figure 4

Figure 4

Autophosphorylation of PKR in infected cells. The Fig is adapted from Ikegami et al.[26] (A) Schematic representations of RVFV S segments of rMP12-NSs-Flag and rMP12-rLuc-Flag. (B) 293 cells were mock-infected or infected with rMP12-NSs-Flag, rMP12-rLuc-Flag or rMP12-PKRΔE7 at an moi of 3, and, then, cells were mock-treated (No drug) or immediately treated with ActD. A cytoplasmic fraction was collected at 16 h.p.i. and the IP-kinase assay of PKR was performed as described previously.[26] A portion of the samples were used for Western blot analysis by using anti-PKR monoclonal antibody to show the abundance of immunoprecipitated PKR (bottom panel).

Figure 5

Figure 5

Analysis of NSs-induced PKR downregulation. The Fig is adapted from Ikegami et al.[26] (A) 293 cells were mock infected (Mock) or infected with MP-12 (MP-12) at an moi of 3. Whole-cell lysates were collected at 2, 4, 6 and 8 h.p.i. Anti-PKR antibody, anti-NSs antibody and anti-β-actin antibody were used to detect PKR, NSs and β-actin, respectively. (B) 293 cells were mock infected (Mock) or infected with MP-12 at an moi of 3 or transfected with in vitro-synthesized RNA transcripts encoding NSs. Cells were then mock-treated or treated with 100μg/ml of puromycin. Cell extracts were harvested at 16 h.p.i. or 16 h post transfection, and the abundance of PKR and viral proteins were analyzed by Western blotting with anti-PKR antibody (top panel), anti-RVFV antibody (middle panel) or anti-β-actin antibody (bottom panel). (C) 293 cells were mock-transfected or transfected with in vitro-synthesized RNA transcripts encoding MP-12 NSs or rLuc. Cells were mock-treated or treated with 5 μg/ml of ActD. Total RNA was harvested at 8 h post transfection, and analyzed by real-time PCR. The relative abundance of PKR mRNA of each sample was calculated by the ΔΔCT method based on the abundance of 18S ribosomal RNA. The data shown in the graph (mean +/- standard deviation) were obtained from three independent experiments. The p value was determined by Student's _t_-test (*: p<0.05). (D) 293 cells were infected with rMP12-rLuc or MP-12 at an moi of 3, and, then, treated with 10 μM of MG132 (MG) or 50 μM of lactacystin (LA) or they were mock treated (-). Whole-cell lysates were collected at 8 h.p.i. and the abundance of PKR, NSs and β-actin was examined by Western blot analysis.

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